The long term goal of this proposal is to determine the roles of SR proteins in trans-splicing. This will be accomplished in an experimental system derived from the parasitic nematode, Ascaris lumbricoides. Our model system offers the unique ability to assay both trans- and cis-splicing in the same extracts in vitro. Additionally, both extracts and splicing factors can be prepared from developmentally staged embryos. The proposed experiments build upon the progress that we have made in establishing the utility of this system in examining the interplay between trans- and cis-splicing as well as its applicability in addressing numerous questions related to the developmental regulation of both SR proteins and alternative splicing. We have also determined that the trans-splicing reaction can be separated into at least two distinct stages allowing us to assay the participation of SR proteins in each step independently. Three specific aims are proposed: 1. Examine the integrated developmental regulation of trans- and cis-splicing. 2. Determine the requirements for SR protein participation in trans-splicing before and after U2 snRNP addition to the substrate. 3. Investigate the role of exon definition and the involvement of U1 snRNP in trans-splicing. A thorough understanding of the trans-splicing reaction may lead to the identification of potential points of therapeutic intervention.